Sepasol rna 1

Isolation of total RNA and quantitative RT-PCR analysis were performed as described previously^{45 (link)}, with minor modifications. In brief, total RNA was prepared from isolated CECs with the use of Sepasol RNA I (Nacalai Tesque) and an RNeasy Mini Kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized from portions (0.8 μg) of the RNA with the use of a QuantiTect Reverse Transcription Kit (Qiagen). The cDNA fragments of interest were amplified by PCR with the use of Fast Start SYBR Green Master (Roche, Penzberg, Germany) and a LightCycler 480 instrument (Roche). The amplification was analyzed with the use of LightCycler 480 software (Roche). The abundance of each target mRNA was normalized by that of hypoxanthine–guanine phosphoribosyltransferase 1 (Hprt1) mRNA. Primer sequences (forward and reverse, respectively) were as follows: Hprt1, 5′-CAGTCCCAGCGTCGTGATTA-3′ and 5′-GGCCTCCCATCTCCTTCATG-3′; Lgr5, 5′-ACCCGCCAGTCTCCTACATC-3′ and 5′-GCATCTAGGCGCAGGGATTG-3′; Ascl2, 5′-CTACTCGTCGGAGGAAAG-3′ and 5′-ACTAGACAGCATGGGTAAG-3′; c-Myc, 5′-CTGGATTTCCTTTGGGCGT-3′ and 5′-TGGTGAAGTTCACGTTGAGGG-3′; Axin2, 5′-GGACTGGGGAGCCTAAAGGT-3′ and 5′-AAGGAGGGACTCCATCTACGC-3′; and cyclin D1, 5′-CAGACGTTCAGAACCAGATTC-3′ and 5′-CCCTCCAATAGCAGCGAAAAC-3'.

Cells were homogenized with Sepasol RNAI (Nacalai Tesque, Kyoto, Japan), and total RNA was isolated following the manufacturer’s instructions. For standard RT-PCR, cDNA was synthesized from 1 μg of total RNA with reverse transcriptase (ReverTra Ace; TOYOBO, Osaka, Japan) and 500 ng of oligo (dT) primer (Life Technologies, Grand Island, NY, USA) for 30 min at 42 °C. A portion of the cDNA was used for real-time PCR. The relative expression of Il-6 gene was determined compared to a reference gene g3pdh using the SYBR^® Green reagent (TaKaRa Bio inc., Kusatsu, Japan). Primers used in these experiments were purchased from TaKaRa, and the sequences were as follows: IL-6: forward primer, 5′- GAGGATACCACTCCCAACAGACC-3′ and reverse primer, 5′- AAGTGCATCATCGTTGTTCATACA-3′; G3PDH: forward primer, 5′- TTCACCACCATGGAGAAGGCCG-3′ and reverse primer, 5′- GGCATGGACTGTGGTCATGA-3′.

Total RNA was isolated from cells treated with each drug using Sepasol-RNA I (Nacalai Tesque) according to the manufacturer’s instructions. Total RNA was reverse transcribed to complementary DNA (cDNA) in a 20 μL reaction volume using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). An equivalent volume of cDNA solution was used for quantitative RT–PCR. cDNA was amplified using a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) with TaqMan Probes for ATF4 (Hs00909569_m1), ASNS (Hs04186194_m1), GLS (Hs00248163_m1), GPT2 (Hs00370287_m1), SLC7A5 (Hs00185826_m1), SLC7A11 (Hs00921938_m1) and β2MG (Hs00984230_m1; Applied Biosystems). The expression of each mRNA was normalized to that of β2MG mRNA in the same sample. All experiments shown were replicated twice.

Total RNA was isolated using Sepasol RNA I (Nacalai Tesque, Kyoto, Japan). mRNA levels were measured as described previously [3 (link)].

Total RNA was isolated from cells treated with each drug or siRNA using Sepasol-RNAI (Nacalai Tesque) according to the manufacturer’s instructions. Total RNA (2 μg) was reversely transcribed to complementary DNA (cDNA) in a 20 μl reaction volume with MMTV-reverse transcriptase (Promega) and oligo (dT) primers (Toyobo, Osaka, Japan). An equivalent volume of cDNA solution was used for quantitative RT–PCR. cDNA was amplified using an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) with TaqMan Probes to CCND1 (Hs00765553_m1) and β2MG (Hs00984230_m1) (Applied Biosystems). The expression of cyclin D1 mRNA was normalized to that of β2MG mRNA in the same sample. All experiments shown were replicated three times.

Plants were grown for 10 days on +S or –S MGRL media. Shoots and roots were harvested separately and divided into 3 replicates, in average 15 plants per replicate. Frozen tissues were mechanically ground to fine powder using Tissue Lyser (Retsch). Total RNA was extracted using Sepasol-RNA I (Nacalai Tesque) followed by the reverse transcription using PrimeScript RT Reagent Kit with gDNA Eraser (Takara). For the reverse transcription, 0.25 µg of the total RNA was used for roots samples. Transcript levels were determined by quantitative real time PCR using KAPA SYBR FAST qPCR Master Mix (2×) kit (Kapa Biosystems, Cape Town, South Africa), and qTOWER³ real-time PCR thermal cyclers (Analytik Jena, Thuringia, Germany) using specific primers (Table S1) [32 (link),70 (link),71 (link)]. Relative expression was calculated by ΔΔCt method with UBQ2 as an internal control. Blank samples were prepared with sterilized distilled water instead of samples. The representative –S responsive genes, BGLU28, SDI1, and SULTR1;1, were analyzed for their transcript levels as a positive control (Figure S2) [39 (link),43 (link)].

RT-PCR and quantitative RT-PCR were performed using RNA isolated from leaves of the three WT (Ldn/PI476874, Ldn/KU-2059, and Ldn/KU-2159) and three chlorosis (Ldn/IG47202, Ldn/KU-2111, and Ldn/KU-20-1) lines. Each plant was grown at 23°C for 3 weeks, and total RNA was extracted using Sepasol-RNA I (Nacalai Tesque, Kyoto, Japan). First-strand cDNA was synthesized from DNase I-treated RNA samples using ReverTra Ace Reverse Transcriptase (Toyobo, Osaka, Japan) and an oligo(dT)₂₀ primer. The gene-specific primer sets for RT-PCR and qRT-PCR are listed in S5 Table. PCR-amplified products were separated by electrophoresis in a 1.5% agarose gel and stained with ethidium bromide. The transcript accumulation of each gene was detected by quantitative RT-PCR using a LightCycler 480 Real-Time PCR System (Roche Diagnostics, Mannheim, Germany) with THUNDERBIRD SYBR qPCR Mix (Toyobo) and gene-specific primer sets. The Actin gene was used as an internal control, and relative expression was calculated as 2^-ΔΔCt, representing the value relative to the transcript levels in leaves of WT (Ldn/PI476874).